1. Field of the Invention
The present invention relates generally to the field of retroviral therapeutic agents and their use in treating retroviral infections and cancers. More specifically it relates to those therapeutic aromatic C-nitroso compounds which destabilize zinc fingers.
2. Description of the Prior Art
The enzyme ADP-ribose transferase (ADPRT) (E.C.4.2.30) is a chromatin.bound enzyme located in the nucleus of most eukaryotic cells. The enzyme catalyzes the polymerization of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD.sup.+) to form poly (ADP-ribose). The polymer is covalently attached to various nuclear proteins, including the polymerase itself.
The many varied roles that ADP-ribosylation plays in cellular metabolism have made ADPRT a target for drugs essentially useful for combating neoplasia and viral infections. Numerous physiological activities have been detected for compounds that inhibit the polymerase activity of ADPRT. Such activities include a cell cycle dependent prevention of carcinogen-induced malignant transformation of human fibroblasts (Kun, E., Kirsten, E., Milo, G. E. Kurian, P. and Kumari, H. L. (1983) Proc. Natl. Acad. Sci. USA 80:7219-7223), conferring also carcinogen resistance (Milo, G. E., Kurian, P., Kirsten, E. and Kun, E. (1985) FEBS Lett. 179:332-336), inhibition of malignant transformation in hamster embryo and mouse C3H10T1/2 cell cultures (Borek, C., Morgan, W. F., Ong, A. and Cleaver, J. E. (1984) Proc. Natl. Acad. Sci. USA 81 243-247), deletion of transfected oncogenes from NIH 3T3 cells (Nakayashu, M., Shima, H., Aonuma, S., Nakagama, H., Nagao. M. and Sugimara, T. (1988) Proc. Natl. Acad. Sci. USA 85:9066-9070), suppression of the mitogenic stimulation of tumor promoters (Roman, F., Menapace, L. and Armato, V. (1983) Carcinogenesis 9:2147-2154), inhibition of illegitimate DNA recombinations (Waldman, B. C. and Waldman, A. (1990) Nucl. Acids Res. 18:5981-5988) and integration (Farzaneh, F., Panayotou, G. N., Bowler, L. D., Hardas, B. D., Broom, T., Walther, C. and Shall, S. (1988) Nucl. Acids Res. 16: 1139-11326), induction of sister chromatid exchange (Ikushima, T. (1990) Chromosoma 99:360-364) and the loss of certain amplified oncogenes (Grosso, L. E. and Pitot, H. C. (1984) Biochem. Biophys. Res. Commun. 119:473-480; Shima, H., Nakayasu, M., Aonums, S., Sugimura, T. and Nagao, M. (1989) Proc. Natl. Acad. Sci. USA 86:7442-7445).
Compounds known to inhibit ADPRT polymerase activity include benzamide (Kun, E., Kirsten, E., Milo, G. E. Kurian, P. and Kumari, H. L. (1983) Proc. Natl. Acad. Sci. USA 80:7219-7223), substituted benzamides (Borek, C., Morgan, W. F., Ong, A. and Cleaver, J. E. (1984) Proc. Natl. Acad. Sci. USA 81:243-247; Romano, F., Menapace, L. and Armato, V. (1983) Carcinogenesis 9; 2147-2154; Farzaneh, F., Panayotou, G. N., Bowler, L. D., Hardas, B. D., Broom, T., Walther, C. and Shall, S. (1988) Nucl. Acids Res. 16:11319-11326.; Grosso, L. E. and Pitot, H. C. (1984) Biochem. Biophys. Res. Commun. 119:473-480; Shima, H., Nakayasu, M., Aonums, S., Sugimura, T. and Nagao, M. (1989) Proc. Natl Acad. Sci. USA 86:7442-7445), 3-aminonaphthylhydrazide (Waldman, B. C. and Waldman, A. (1990) Nucl. Acids Res. 18:5981-5988), isoquinoline, quercetin, and coumarin (1,2-benzopyrone) (Milo, G. E., Kurian, P., Kirsten, E. and Kun, E. (1985) FEBS Lett. 179: 332-336). The antitransforming and anti-neoplastic effect of 1,2 benzopyrone were demonstrated in vitro and in vivo (Tseng, et al., (1987) Proc. Natl. Acad. Sci. USA 84:1107-1111).
The 6-nitroso-benzopyrones were only recently discovered (U.S. patent application No. 07/412,783). The only remotely related compounds found in the literature are 6-nitro-1,2-benzopyrone and 6-amino-1,2-benzopyrone (6-ABP) (J. Pharm. Soc. Jap., 498:615 (1923)) for which, only scarce medicinal evaluation has been reported. In particular, testing was done for sedative and hypnotic effects (J. Pharm. Soc. Japan. 73:351(1953); Ibid. 74:271(1954)), hypothermal action (Yakuoaku Zasshi. 78:491 (1958)), and antipyretic, hypnotic, hypotensive and adrenolytic action (Ibid. 83:1124 (1963)). No significant application for any of these compounds has been described except for 6-ABP.
2-nitrosobenzamide (Irne-Rasa, K. M. and Koubek, E. (1963) J. Org. Chem. 28:3240-3241), and 4-nitrosobenzamide (Wubbels, G. G., Kalhorn, T. F., Johnson, D. E. and Campbell, D. (1982) J. Org. Chem. 47:4664-4670), have been reported in the chemical literature, but no medical or therapeutic use of these isomers is known. Neither of these articles suggest the use of nitrosobenzamides as ADPRT inhibitors or for use as anti-retroviral agents or 10 anti-cancer agents.
The anti-viral and anti-tumorigenic actions of substituted and unsubstituted 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone is the subject of copending U.S. patent applications Ser. No 585,231 filed on Sep. 21, 1990 entitled "6-Amino-1,2-Benzopyrones Useful for Treatment of Viral Diseases" and Ser. No. 600,593 filed on Oct. 19, 1990 entitled "Novel 5-Iodo-6-Amino-1,2-Benzopyrones and Their Metabolites Useful as Cytostatic and Antiviral Agents", which are incorporated herein by reference.
The precursor molecule, 1,2-benzopyrone (coumarin), was shown to be an inhibitory ligand of adenosinediphosphoribosyl transferase (ADPRT), a DNA-binding nuclear protein present in all mammalian cells (Tseng, et al, (1987) Proc. Nat. Acad. Sci. USA. 84:1107-1111).
Hakam, et al., FEBS Lett., 212:73 (1987) has shown that 6-amino-1,2-benzopyrone (6-ABP) binds specifically to ADRPT at the site that also binds to DNA, indicating that both 6-ABP and DNA compete for the same site on ADPRT. Synthetic ligands of ADPRT inhibit DNA proliferation, particularly in tumorigenic cells, (Kirsten, et al., (1991) Exp. Cell. Res. 193:1-4). These ligands have been found to inhibit viral replication and are the subject of the copending U.S. patent application entitled "6-Amino-1,2-Benzopyrones useful for Treatment of Viral Diseases," Ser. No. 585,231, filed on Sep. 21, 1990 which is hereby incorporated by reference.
Retroviral nucleocapsid (NC) proteins and their respective gag precursors from all strains of known retroviruses contain at least one copy of a zinc-binding polypeptide sequence of the type Cys-X.sub.2 -Cys-X.sub.4 -His-X.sub.4 -Cys (CCHC) (Henderson, et al., Biol. Chem. 256:8400-8406 (1981)), i.e., a zinc finger domain. This CCHC sequence is essential for maintaining viral infectivity (Gorelick, et al., Proc. Natl. Acad. Sci. USA 85:8420-8424 (1988), Gorelick, et al., J. Virol. 64:3207-3211(1990)), therefore, it represents an attractive target for viral chemotherapy. The HIV-1 gag proteins function by specifically binding to the HIV-1 RNA, anchoring it to the cell membrane for budding or viral particles (Meric, , et al., J. Virol. 63: 1558-1658 (1989) Gorelick, et al., Proc. Natl. Acad. Sci. USA 85:8420-8424 (1988), Aldovini, et al., J. Virol. 64:1920-1926 (1990), Lever, et al, J. Virol. 63:4085-4087 (1989)). Site-directed mutagenesis studies demonstrated that modification of Cys or His residues results in defective viral RNA packaging and noninfectious viral particles are formed (Aldovini et al., J. Virol. 64:1920-1926 (1990), Lever, et al, J. Virol. 63:4085-4087 (1989)). The highly abundant nonhistone nuclear protein of eukaryotes, poly(ADP-ribose) polymerase (E.C.2.4.4.30), also contains two CCHC-type zinc fingers located in the basic terminal polypeptide domain, as analyzed by site directed mutagenesis (Gradwohl, et al., Proc. Natl. Sci. USA 87:2990-2992 (1990)).
In copending U.S. Ser. No. 07/893,429 filed Jun. 4, 1992 the disclosure of which is incorporated herein by reference, it is shown that 3-nitrosobenzamide and 6-nitroso-1,2-benzopyrone inhibit propagation of HIV-1 in human lymphocytes and also eject zinc from isolated HIV-1 NC fingers.
Recently published experiments have shown that aromatic C-nitroso ligands of poly (ADP-ribose) polymerase preferentially destabilize one of the two zinc fingers coincidental with a loss of enzymatic activity but not DNA binding capacity of the enzyme protein (Buki, et al., FEBS Lett. 290:181-185 (1991)). Based on the similarity to results obtained by site-directed mutagenesis (Gradwohl, et al, Proc. Natl. Sci. USA 87:2990-2992 (1990)), it appears that the primary attack of C-nitroso ligands occurred at zinc finger FI (Buki, et al., FEBS Lett. 290:181-185 (1991)). 6-nitroso-1,2-benzopyrone (6-NOBP) and 3-nitrosobenzamide (3-NOBA), two C-nitroso compounds that inactivate ADPRT at one zinc finger site completely suppressed the proliferation of leukemic and other malignant human cells and subsequently produced cell death. Tumoricidal concentrations of the drugs were relatively harmless to normal bone marrow progenitor cells and to superoxide formation by neutrophil granulocytes. The cellular mechanisms elicited by the C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesium dependent endonuclease (Rice et al. Proc. Natl. Sci. USA (1992) 89:7703-7707. This endonuclease is maintained in a latent form by poly(ADP-ribosyl)ation, but inactivation of ADPRT by C-nitroso drugs depresses the DNA-degrading activity.
While these nitroso compounds have been found to be quite effective in preliminary tests, they are relatively water insoluble at physiological pH, exhibit limited stability and limited predictability of delivery to the affected cells due to their solubility and stability characteristics. Thus it is of interest to construct C-nitroso compounds, especially the aromatic C-nitroso compounds, in a more stable and more water soluble form for convenient use as anti-retroviral and anti-cancer compositions.
The chemical reaction of an aromatic sulfinic acid (p-tolunesulrinic acid) with an Ar--N.dbd.O (nitrosobenzene) is known (Chemische Berichte. 34: 228-241 (190I). The preparation and use of aromatic sulfinic acids (specifically benzenesulfinic acid and p-toluenesulfinic acid) as chemical protecting groups for certain Ar--N.dbd.O compounds (e.g., p-PhC(O)--C.sub.6 H.sub.4 -- N.dbd.O) for purposes of electrochemical synthesis has been reported J. C. S. Chem Commun., 820 (1976). The method was developed for trapping and protecting nitroso compounds generated during electrochemical synthesis. No suggestion was made that these compounds could be used as drugs.
The preparation of adducts of alkyl--N.dbd.O compounds with aryl sulfinic acids (Rec.Trav.Chim. 89:696-704 (1970) has been reported as well as the reaction of simple alkyl sulfinic acids with Ar--N.dbd.O compounds (Chem. Abstr. 89:189390s (1977). The use a sulfinic acid derivative of the amino acid (L-cysteine-sulfinic acid, denoted as L-alanine,2-sulfino in Chemical Abstracts) has not been reported, however.